Fig. 3

Cilia structure and function are altered in human LUHMES RFX2 -/- mutants. See also Additional file 2: Fig. S1. A Schematic illustration of the RFX2 transcription factor (TF) gene structure with functional protein domains indicated: AD – activation domain, DBD – DNA binding domain, B and C – domains B and C of unknown function, DIM – dimerization domain. CRISPR/Cas9-engineered, small deletions lead to reading frame shifts upstream of the RFX2 DBD, essential for TF functionality, thereby creating an RFX2 -/- knockout mutant. B Western blot analyses of RFX2 protein expression in LUHMES wild-type (WT) and RFX2 -/- mutants: In the RFX2 -/- mutant RFX2 protein expression is completely absent during early neuron differentiation time points (d1-d3) and at the end of differentiation and maturation (d6). Also, no protein expression of a hypothetical, predicted truncated RFX2 protein product was detectable because of CRISPR/Cas9 mutagenesis. The housekeeping protein GAPDH was used as a loading control. C WT (n = 171) and RFX2 -/- (n = 215) neuronal populations display a similar and uniform distribution of cilia orientation in 3D space. D Cilia of RFX2 -/- mutants are longer than WT cilia in differentiating neurons (d3). Immunofluorescence-based length measurements: ARL13B marks the ciliary shaft, PCNT marks the basal body. Statistics: WT (n = 76), RFX2 -/- (n = 111). E Representative immunocytochemistry images of WT vs elongated RFX2 -/- cilia. F Ciliary signaling pathway genes relevant for neuron differentiation are deregulated between differentiating WT and RFX2 -/- neurons. Mean values ± s.e.m. are shown from two independent experiments with a minimum of three technical replicates each. We conducted regular one-way ANOVA (D) and two-way ANOVA analyses (C, F) (not repeated measures) with multiple comparisons (Bonferroni’s test) between groups. *p < 0.05; **p < 0.005; ***p < 0.0005; ****p < 0.0001